Retinyl ester formation by lecithin: retinol acyltransferase is a key regulator of retinoid homeostasis in mouse embryogenesis.

The developing mammalian embryo is entirely dependent on the maternal circulation for its supply of retinoids (vitamin A and its metabolites). The mechanisms through which mammalian developing tissues maintain adequate retinoid levels in the face of suboptimal or excessive maternal dietary vitamin A intake have not been established. We investigated the role of retinyl ester formation catalyzed by lecithin:retinol acyltransferase (LRAT) in regulating retinoid homeostasis during embryogenesis. Dams lacking both LRAT and retinol-binding protein (RBP), the sole specific carrier for retinol in serum, were maintained on diets containing different amounts of vitamin A during pregnancy. We hypothesized that the lack of both proteins would make the embryo more vulnerable to changes in maternal dietary vitamin A intake. Our data demonstrate that maternal dietary vitamin A deprivation during pregnancy generates a severe retinoid-deficient phenotype of the embryo due to the severe retinoid-deficient status of the double mutant dams rather than to the lack of LRAT in the developing tissues. Moreover, in the case of excessive maternal dietary vitamin A intake, LRAT acts together with Cyp26A1, one of the enzymes that catalyze the degradation of retinoic acid, and possibly with STRA6, the recently identified cell surface receptor for retinol-RBP, in maintaining adequate levels of retinoids in embryonic and extraembryonic tissues. In contrast, the pathway of retinoic acid synthesis does not contribute significantly to regulating retinoid homeostasis during mammalian development except under conditions of severe maternal retinoid deficiency.

Pathways of vitamin A delivery to the embryo: insights from a new tunable model of embryonic vitamin A deficiency.

Circulating retinoids (vitamin A and its derivatives) are found predominantly as retinol bound to retinol-binding protein (RBP), which transports retinol from liver stores to target tissues, or as retinyl ester incorporated in lipoproteins of dietary origin. The transport of retinoids from maternal to fetal circulation is poorly understood, especially under conditions of inadequate dietary vitamin A intake. Here we present RBP-/- mice as a tunable model of embryonic vitamin A deficiency. This model has enabled us to analyze metabolic links between maternal nutrition and retinoid delivery to the fetus. Our data show that retinol-RBP is the primary contributor to fetal development, whereas retinyl ester are largely responsible for accumulation of fetal retinoid stores. Furthermore, these studies indicate the importance of embryonic RBP in distributing vitamin A to certain developing tissues under restrictive diets. We also show differences among developing tissues in their dependency on the embryonic retinol-RBP pathway. Finally, we demonstrate that accumulation of embryonic vitamin A stores does not depend on the expression of RBP in the fetal liver.

The role of extrahepatic retinol binding protein in the mobilization of retinoid stores.

Although the major tissue site of retinol binding protein (RBP) synthesis in the body is the liver, other sites of synthesis have been reported. The physiological role(s) of circulating RBP that is produced and secreted extrahepatically has not been systematically investigated. To address this question, we used as a model a mouse strain (hRBP(-/-)) that expresses human RBP (hRBP) cDNA under the control of the mouse muscle creatine kinase promoter in an rbp-null background (RBP(-/-)). By comparing hRBP(-/-), RBP(-/-), and wild-type mice, we asked whether extrahepatic RBP can perform all of the physiological functions of RBP synthesized in the liver. We demonstrate that extrahepatically synthesized hRBP, unlike RBP expressed in liver, cannot mobilize liver retinoid stores. Consistent with this conclusion, we find that circulating hRBP is not taken up by hepatocytes. RBP has been proposed to play an essential role in distributing hepatic retinoids between hepatocytes and hepatic stellate cells. We find, however, that the distribution of retinoid in the livers of the three mouse strains described above is identical. Thus, RBP is not required for intrahepatic transport and storage of retinoid. These and other observations are discussed.

Vitamin A: overlapping delivery pathways to tissues from the circulation.

Although retinol bound to retinol-binding protein (RBP) is the most abundant retinoid form present in the circulations of humans and most mammals, other retinoid and proretinoid forms are also present in the blood. We are interested in understanding to what extent each of these circulating retinoid forms contributes towards retinoid actions within cells and tissues. Here we report two studies focused on this question. First, we examined retinoid transport and storage in RBP-deficient mice that lack circulating RBP. These mice under normal laboratory conditions are phenotypically normal except for a visual impairment early in life that is corrected if the mice are maintained on a vitamin A-sufficient diet throughout life. The RBP-deficient mice take up vitamin A from the diet into most tissues at least as well as wild type mice. Compared to wild type mice, mice lacking RBP accumulate excess vitamin A in the liver, since there is no RBP to facilitate mobilization of stored retinol from hepatic stores. In a second study, we explored in vitro the actions of carotene cleavage enzyme (CCE) in facilitating beta-carotene cleavage to retinoid in the testis. CCE is most highly expressed in the testis. Pull-down experiments coupled with MALDI-MS analysis showed that mouse testis CCE is able to interact with the testis-specific lactate dehydrogenase-C (LDH-C) isoform. This may suggest that CCE and LDH-C act in concert to catalyze beta-carotene cleavage.

Transplacental delivery of retinoid: the role of retinol-binding protein and lipoprotein retinyl ester.

Retinoids are required for normal embryonic development. Both embryonic retinoid deficiency and excess result in congenital malformations. There is little understanding of the physiology underlying retinoid transfer from the maternal circulation to the embryo. We now report studies that explore this process using retinol-binding protein-deficient (RBP-/-) mice and mice that express human RBP on the RBP-/-) background. Our studies establish that dietary retinoid, bound to lipoproteins, can serve as an important source for meeting tissue retinoid requirements during embryogenesis. Indeed, retinyl ester concentrations in the circulations of pregnant RBP-/- mice are significantly elevated over those observed in wild-type mice, suggesting that lipoprotein retinyl esters may compensate for the absence of retinol-RBP during pregnancy. We also demonstrate, contrary to earlier proposals, that maternal RBP does not cross the placenta and cannot enter the fetal circulation. Overall, our data indicate that both retinol-RBP and retinyl esters bound to lipoproteins are able to provide sufficient retinoid to the embryo to allow for normal embryonic development.

Understanding the physiological role of retinol-binding protein in vitamin A metabolism using transgenic and knockout mouse models.

Retinoids (vitamin A and its derivatives) play an essential role in many biological functions. However mammals are incapable of de novo synthesis of vitamin A and must acquire it from the diet. In the intestine, dietary retinoids are incorporated in chylomicrons as retinyl esters, along with other dietary lipids. The majority of dietary retinoid is cleared by and stored within the liver. To meet vitamin A requirements of tissues, the liver secretes retinol (vitamin A alcohol) into the circulation bound to its sole specific carrier protein, retinol-binding protein (RBP). The single known function of this protein is to transport retinol from the hepatic stores to target tissues. Over the last few years, the generation of knockout and transgenic mouse models has significantly contributed to our understanding of RBP function in the metabolism of vitamin A. We discuss below the role of RBP in maintaining normal vision and a steady flux of retinol throughout the body in times of need.

Muscle expression of human retinol-binding protein (RBP). Suppression of the visual defect of RBP knockout mice.

Mice lacking retinol-binding protein (RBP) have low circulating retinol levels. They have severe visual defects due to a low content of retinol or retinyl esters in the eye. A transgenic mouse strain that expresses human RBP under the control of the muscle creatine kinase promoter in the null background was generated. The exogenous protein bound retinol and transthyretin in the circulation and effectively delivered retinol to the eye. Thus, RBP expressed from an ectopic source suppresses the visual phenotype, and retinoids accumulate in the eye. No human RBP was found in the retinal pigment epithelium of the transgenic mice, indicating that retinol uptake by the eye does not entail endocytosis of the carrier RBP.